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Components of the Bio-Rad Silver Stain Plus (SS+) Process n Fixative enhancer solution n H 2 O purified by filtration through ion-exchange resins and organic trapping resins n Staining solution n Stop solution There is more about each of these on the following slides.

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Components of the SS+ Process Staining solution (cont’d) n Silver complex solution (AgNO 3 and NH 4 NO 3 ) u provides the Ag+ that is reduced to Ag u just over 2 NH 4 NO 3 to 1 AgNO 3 n Reduction moderator solution u H 2 O solution of dodecatungstosilicic acid (TSA), a heteropoly acid (H 4 O 40 SiW 12 ) F Similar to an ion exchange bead F Serves as carrier for the Ag+ complex so that it is not free in solution. This is important!!

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Components of the SS+ Process Staining Solution (cont’d) n Image developer reagent (formaldehyde) u Formaldehyde is a reducing agent in alkaline conditions. u It is oxidized to formic acid. u It will not reduce Ag + bound to tungstosilicic acid as rapidly as it will reduce unbound ions.

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After the mixed staining solution is applied to the gel..... n At first, almost no metallic silver is formed u at the locations of DNA or protein u in solution u in the gel matrix n But, nucleophilic, aromatic, and heteroaromatic groups in proteins and DNA u readily compete with TSA to form complexes with Ag + and u Ag + in complex with DNA or protein is not protected from reduction by formaldehyde

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So... n Silver complexed with DNA or protein is rapidly reduced n An autocatalysis occurs u Ag facilitates reduction of nearby Ag+ n And after a while......, as if out of nowhere....., bands appear with almost no background ! ! !

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Imagine.... n The incredible balance required between u The acidity of the TSA and its buffering capacity vs. u The need to have carbonate create basic conditions to F activate the formaldehyde F deprotonate the NH4 +

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Success Precautions n Prevent Ag 2 CO 3 precipitate by adding Na 2 CO 3 quickly and all at once. u Adding it slowly prevents formation of NH 3 and the NH 3 /Ag+ complex and allows free Ag+ to precipitate with the excess of CO 3 = u If too much precipitate is allowed to form, it may not go back into solution. u The Ag + in the precipitate is readily reduced  background from autocatalysis. The precipitate is sticky.

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Success Precautions n Be sure the gel is submerged and moving freely during exposure to all solutions. n Avoid orbital, as opposed to oscillating, shakers. Orbital shakers  vortex, which causes concentration gradients to develop from the central portion of the gel to the outside. n Avoid microheterogeneities in the surface of the dish or gel. They trigger the reduction chemistry. u cracks and compressions in the gel u scratches in the container

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Success Precautions (cont’d) n Use freshly made CO 3 = solutions. u carbonate solutions tend to dissolve CO 2 from the air  HCO 3 - which causes any precipitates which form to be more problematic. n Avoid conjugated unsaturations; they complex with Ag+ u found in polyester, polycarbonate, plasticized (flexible) polypropylene or polyethylene F Plasticizers are often esters of phthallic acid, which is aromatic, and so has conjugated unsaturations.

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Thanks to Bio-Rad tech support for providing details of the reaction not included with the kit!!!!